Molecular basis for determining the sensitivity of eucaryotes to the antimitotic drug rhizoxin.
Rhizoxin, an antibiotic, exhibits potent anti-mitotic activity against most eucaryotic cells including those of higher vertebrates, plants and fungi by binding to beta-tubulin. The benA gene of three independently isolated rhizoxin-resistant (Rhir) mutants of Aspergillus nidulans was cloned, sequenced and compared with that of the wild-type, rhizoxin-sensitive (Rhis) strain. In all three Rhir mutants, the AAC codon for Asn-100 of the benA beta-tubulin gene was altered to ATC, coding for Ile. Sequence displacement experiments confirmed that the substitution of Ile for Asn-100 confers resistance to rhizoxin in this organism. The amino acid sequences of beta-tubulin surrounding the 100th amino acid residue from the N-terminus including Asn-100 are highly conserved with a few exceptions. The fission yeast Schizosaccharomyces pombe and the budding yeast Saccharomyces cerevisiae are naturally occurring Rhir organisms whose beta-tubulin genes encode Ile and Val respectively at the 100th amino acid residue. The Ile-100 of S. pombe and the Val-100 of S. cerevisiae were altered to Asn using site-directed mutagenesis and gene displacement techniques. The resultant haploid strains of these two yeasts uniquely expressing beta-tubulin (Asn-100) instead of beta-tubulin (Ile-100 or Val-100) were found to be Rhis. Haploid yeast expressing beta-tubulin (Asn-100) is normal except for its sensitivity to rhizoxin. These results suggest that rhizoxin resistance has a common basis in both naturally occurring species and experimentally selected mutants in the substitution of Ile or Val for Asn-100 in beta-tubulin.
Takahashi
M
M
Institute of Applied Microbiology, University of Tokyo, Japan.
Matsumoto
S
S
Iwasaki
S
S
Yahara
I
I
D000595:Amino Acid Sequence; D000935:Antifungal Agents / Q000494:pharmacology*; D001233:Aspergillus nidulans / Q000187:drug effects* / Q000235:genetics; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D003062:Codon; D004271:DNA, Fungal; D004352:Drug Resistance, Microbial / Q000235:genetics; D005800:Genes, Fungal; D007783:Lactones / Q000494:pharmacology; D018942:Macrolides; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D009154:Mutation; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics; D012568:Schizosaccharomyces / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D014170:Transformation, Genetic; D014404:Tubulin / Q000235:genetics*
D000935:Antifungal Agents; D003062:Codon; D004271:DNA, Fungal; D007783:Lactones; D018942:Macrolides; D014404:Tubulin; C041443:rhizoxin